This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mitogen-activated protein kinase (MAPK) signaling cascades play a fundamental role in eukaryotic cells, acting as signal transducers involved in many biological processes including growth, differentiation and response to stress. In yeast, the mating, filamentation, and high osmolarity glycerol (HOG) pathways elicit specific responses to distinct stimuli, yet share multiple components. We observed that both the MEK and the MAPKs of the mating and filamentation pathways are phosphorylated on their activating residues during signaling through the HOG pathway. Therefore, mechanisms must exist downstream of the MAPKs to ensure that mating and filamentation genes are not transcribed in response to osmotic stress. In addition, we found that this is a novel mechanism, distinct from the mechanisms used to prevent cross-talk between the filamentation and mating pathways. We tested whether specificity is achieved before or after the mating and filamentation specific transcription factors, Ste12 and Tec1, are recruited to their gene targets, and found that in the absence of Hog1 there is a large increase in the levels of Tec1 binding to filamentation specific promoters. This indicates that Hog1 plays an essential role in inhibiting erroneous signaling by preventing Tec1 from binding to its target promoters. These results point to new modes of regulation used by eukaryotic cells to perform the indispensable function of maintaining signaling specificity.